DESCRIPTION (Adapted from the investigator's application): Voltage-gated Ca2+ channels do a remarkable job of efficiently catalyzing the downhill flow of Ca2+ into cells, allowing Ca2+ permeation with rapid turnover rates, while also showing exquisite selectivity for Ca2+ over more abundant extracellular ions like Na+. A single opening of a Ca2+ channel may allow many thousands of Ca2+ ions to enter the cytoplasm generating a rise in [Ca2+]i that can control transmitter release, excitability, metabolism of gene expression. The structural determinants of Ca2+ channel selectivity and ion permeation are only partially understood. A set of four glutamate residues, located at a homologous position in each of the four repels of Ca2+ channel a1 subunits, lie within the pore and display high-affinity interactions with Ca2+ and other divalent cations. In this project, new experiments will be undertaken to gain more information about the nature of the Ca2+ channel pore and outer vestibule, and to clarify the mechanism by which the pore glutamates and other structures support ion permeation and block. The molecular determinants of the cut-off size of the pore's "selectivity filter" and its permeability to internal ions will be studied. The outer vestibule of channel will be systematically probed using variants of conesnail peptide toxins as molecular calipers. Stations between bound toxin and permeating ions or channel inactivation will be examined in an effort to know more about external conformational changes during channel gating. Various configurations of the channel such as its protonated and Ca2+-blocked states will be incorporated into an increasingly sophisticated series of permeation models that will take account of the dimensions of the pore and the amino acid side chains within it. Kinetic features of permeation and block will also be modeled. Information about the structure of the pore will not only clarify the mechanism of permeation, but will also help explain the pore's ability to undergo high-affinity, Ca2+-dependent interactions with drugs such as 1,4-dihydropyridines.